Melbourne Veterinary School - Theses
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ItemPhylogeny and virulence factor genes of canine urinary Escherichia coli in relation to clinical disease and antimicrobial resistance( 2018)Traditionally, urinary tract infections (UTIs) have been categorised as either uncomplicated or complicated in veterinary medicine, with treatment differing for the two categories. In human medicine, there is an additional category: asymptomatic bacteriuria, which is the presence of bacteriuria without symptoms of infection. Escherichia coli is the most common bacterial species involved in UTIs in dogs. Clinical signs can be absent in dogs with complicated UTIs, and this has been likened to asymptomatic bacteriuria in people and has been termed subclinical bacteriuria (SBU). Treatment recommendations for SBU in dogs have been adapted from human recommendations. Many E. coli strains are resistant to multiple antibiotics and uropathogenic E. coli possess virulence factor genes that facilitate overcoming host defence mechanisms. These E. coli commonly belong to phylogenetic groups B2 and D. Some studies suggest that human E. coli isolated from asymptomatic bacteriuria differ from those causing clinical UTI. While the virulence factor genes and phylogeny of canine urinary E. coli isolated from UTIs are well characterised, little is known about virulence factor genes and phylogeny in E. coli isolated from SBU. Furthermore, these genomic characteristics have not been studied in detail in multi-drug resistant (MDR) canine urinary E. coli. Thus, the pathogenic potential of canine urinary E. coli is not well described and the benefit of antibiotic therapy in SBU and MDR infections is unknown. This study used whole genome sequencing to characterise 47 E. coli isolated from dogs with SBU and 67 E.coli from dogs with clinical UTI in terms of their phylogeny and virulence factor gene profile. From those strains, the 15 MDR strains were characterised. When the phylogeny and virulence factor gene profile of E. coli isolated from dogs with SBU were compared with clinical UTIs, results showed that most clinical UTI and SBU E. coli belonged to phylogenetic group B2. The virulence factor gene profile was similar between the two groups, and no association was found between them and the 83 virulence factor genes analysed. Many of the MDR E. coli belonged to phylogenetic group B1 and these isolates possessed fewer virulence factor genes than non-MDR E. coli. Based on the results of this study, it was concluded that phylogeny and the presence of virulence factor genes do not influence the manifestation of clinical disease. Host immunity and rather than presence and the expression of or mutations of virulence factor genes may have a role in the development of clinical disease. ii MDR E. coli have fewer virulence factor genes than non-MDR isolates, with MDR isolates commonly belonging to commensal phylogenetic groups. This suggests that treatment of MDR isolates is not always indicated because they tend to have commensal bacterial phylogeny, which can have implications for reducing the development of antimicrobial resistance. While whole genome sequencing is an accurate method for determining phylogeny and the presence of virulence factors, it is unable to easily differentiate the pathogenicity potential of urinary E. coli and therefore treatment recommendations cannot be made based on this technique.
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ItemAssessment of a ropivacaine ultrasound-guided Transversus Abdominis Plane (TAP) block for peri-operative analgesia in the dog undergoing ovariohysterectomy surgery( 2019)Advances in veterinary medicine have led to the development of further techniques to provide analgesia for our patients. Ovariohysterectomy is a commonly performed surgery in female dogs which causes significant pain. Multimodal analgesia often combining opioids, non-steroidal anti-inflammatories and local anaesthetic techniques is implemented to treat this type of pain. The transversus abdominis plane (TAP) block is a local anaesthetic technique that provides analgesia to the afferent nerves supplying the parietal peritoneum, muscles and skin of the anterior abdominal wall. The TAP block is utilized in human anaesthesia and has been shown to reduce post-operative pain scores and opioid consumption. The TAP block has potential to provide local anaesthesia to the mid-caudal abdomen for dogs undergoing ovariohysterectomy. The initial pilot study was a cadaveric study carried out in 5 dogs with an ultrasound guided two-point TAP injection performed in each hemiabdomen. The study identified that a two-point TAP injection delivered consistent dye dispersion to adequately stain branches of thoracic nerve T13 and lumbar nerves L1, L2 and L3. It concluded that this technique should be assessed in vivo to evaluate the analgesic efficacy in mid to caudal abdominal surgeries. A subsequent randomized controlled trial assessed the two-point ultrasound-guided ropivacaine TAP block for dogs undergoing ovariohysterectomy, with the hypothesis that dogs receiving the ropivacaine TAP block would have a lower minimum alveolar concentration (MAC) isoflurane at skin incision and a lower opioid rescue analgesic requirement post-operatively. The results concluded that the estimated population MACiso for the treatment group was lower than the control group but that this was not significant. There was also no significant difference in pain scores between the groups as measured by the Glasgow composite pain scale short form and mechanical nociceptive threshold testing. The proportion of subjects receiving rescue analgesia in the treatment group was lower than the control group, but this was not significant. The study concluded that the two-point TAP block did not provide significant MAC reduction or additional intra-operative or post-operative analgesia compared with the control for dogs undergoing ovariohysterectomy. Multiple confounding factors such as the administration of morphine as premedication and the low concentration of local anaesthetic used, ropivacaine 0.2%, could have contributed to these findings. Although the cadaver study identified that a two-point TAP injection delivered consistent dye dispersion to thoracic nerve T13 and lumbar nerves L1, L2 and L3, the technique when performed with 0.2% ropivacaine in dogs undergoing ovariohysterectomy did not provide significant additional MACiso reduction or intra-operative or post-operative analgesia compared with the control for dogs. Further assessment of the toxic dose of ropivacaine in dogs and the minimum effective concentration of ropivacaine for TAP blocks in dogs needs to be evaluated in order to further develop and assess this technique.
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ItemInflammation and endothelial perturbation in canine abdominal surgery: the potential modulatory effect of lidocaine( 2019)Complication rates following emergency laparotomy surgery are high, with organ dysfunction being a commonly encountered post-operative complication. Given the endothelium acts as the interface between the systemic circulation and the organs, its function is vital to maintaining organ health. The endothelium is in a constant state of flux, impacted largely by the local environment of which it is a part. In the presence of wide-spread systemic inflammation, inflammatory mediators precipitate change to the structure of the endothelial glycocalyx. These changes result in shedding of the endothelial glycocalyx and alteration of the endothelial phenotype. The endothelium may, as a result, lose the capacity to regulate vasomotor tone, and shift toward a pro-inflammatory and pro-coagulant state. This predisposes to reduced tissue oxygen delivery, and organ dysfunction may ensue. This thesis aimed to answer two key questions: does surgical trauma induced in canine patients undergoing emergent abdominal surgery invoke a systemic inflammatory response and subsequent endothelial activation? And if so, does lidocaine, a proposed immunomodulatory drug, mitigate this effect when given in the post-operative period? Chapter two provides a detailed review of endothelial structure and function, and current literature pertaining to systemic inflammation and endothelial activation in the context of abdominal surgery. Chapter two also examines the literature regarding the proposed mechanisms through which lidocaine acts as an immunomodulatory drug, and reviews publications that investigate the use of lidocaine as an anti-inflammatory drug in human patients after abdominal surgery. Chapter three is a randomized, blinded clinical trial quantifying the effect of emergency abdominal surgery on the concentration of markers of systemic inflammation and endothelial perturbation in canine patients in the post-operative period. The trial also assessed the potential use of lidocaine as a post-operative immunomodulatory therapy in dogs having undergone laparotomy. Fifty canine patients undergoing abdominal surgery were enrolled in the study. Patients were randomized into two separate groups: a study group receiving lidocaine intravenously, and a control group receiving 0.9% NaCl intravenously for a twelve-hour period following abdominal surgery. Blood samples were gathered prior to surgery, followed by six and twelve hours post-operatively. Concentrations of markers of systemic inflammation (IL-6) and markers of endothelial perturbation (VEGF and HA) were quantified via means of ELISA at each time point. Results revealed a significant increase in the concentration of markers of systemic inflammation and endothelial perturbation in post-operative blood samples. No immunomodulatory or endothelial preserving effect of lidocaine was appreciated.
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ItemInvestigating the inflammatory pathways involved in placental detachment in the mare( 2018)The mechanism of placental detachment in the mare has not been investigated in detail and as such remains poorly understood. Research in other species provides strong evidence that parturition and fetal membrane detachment from the endometrium requires involvement of the immune response. Research in horses has also suggested that movement of leukocytes in the umbilical vessels, and their attraction to fetal membranes at parturition, contribute to placental detachment. The aims of this study were to 1) confirm whether a leukocyte “concentration gradient” exists between the equine neonatal umbilical artery and vein, and whether this gradient was associated with fetal membrane retention time 2) to investigate the expression of pro-inflammatory cytokines in the equine endometrium and fetal membranes at parturition using quantitative real time polymerase chain reaction (RT-qPCR) and 3) to localise pro-inflammatory cytokine proteins within the equine endometrial and fetal membrane tissues using immunohistochemistry to correlate with gene expression. Data was collected from 33 spontaneously foaling mares and their foals on a single Thoroughbred stud farm in New Zealand. Umbilical artery and vein blood, fetal membrane and endometrial biopsy tissue samples were obtained. The average fetal membrane retention time was 92 (+/-130) minutes. The median fatal membrane retention time was 47 minutes (IQR 30). There was a significantly higher number of total leukocytes, lymphocytes and neutrophils in the equine neonatal umbilical artery than the umbilical vein at parturition (p <0.001). Fetal membrane retention time tended to decrease with an increase in umbilical leukocyte concentration gradient but did not reach statistical significance (p = 0.098). The mRNA expression of IL-1, IL-6 and IL-8 was significantly greater in the endometrium of foaling mares compared to control mares (p < 0.001). The mRNA expression of IL-8 was significantly higher in the fetal membranes of foaling compared to control mares (p < 0.001) whereas mRNA expression of IL-6 in fetal membranes was significantly lower between the two groups (p< 0.001). Whilst mRNA expression for IL-1 was lower in the fetal membranes of foaling mares compared to controls, the difference was not significant (p = 0.16). This study has demonstrated the presence of an innate inflammatory immune response in the placenta of spontaneously foaling mares, suggesting the role of inflammation in the detachment of the fetal membranes from the endometrium. The results from this study have highlighted the need to further investigate the mechanism of placental detachment in the mare and also understand the pathophysiology behind abnormal fetal membrane retention.
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ItemInvestigation of the risk of Toxoplasma gondii to the establishment of the ‘extinct in the wild’ eastern barred bandicoot (Perameles gunnii) on Phillip Island.( 2018)The eastern barred bandicoot, Perameles gunnii, is currently considered extinct in the wild, and is extant only as intensively managed reintroduced populations. Phillip Island, in Westernport Bay, Victoria, has been proposed as a potential reintroduction site for this species. Feral cats, Felis catus, the definitive host for the coccidian parasite Toxoplasma gondii, are present on the island, and eastern barred bandicoots are known to be susceptible to toxoplasmosis. The aim of this study was to investigate the epidemiology of T. gondii on Phillip Island, and the potential risk to the establishment of eastern barred bandicoot populations. The prevalence of T. gondii in the feral cat population on Phillip Island was determined using real time PCR (qPCR), and seroprevalence was determined using the modified agglutination test (MAT). A total of 161 feral cats was sampled between June 2016 and November 2017. Overall prevalence by qPCR was 79.5 % (95 % confidence interval [95% CI] 72.6 - 85.0), and overall seroprevalence was 91.8 % (95% CI 84.6 – 95.8). Additionally, the diagnostic sensitivity and specificity of both testing methods, and the true prevalence of T. gondii in feral cats on Phillip Island, were evaluated using Bayesian modelling. The sensitivity and specificity of the MAT in cats were estimated as 96.1 % (95 % credible interval [95% CrI] 91.5 – 98.8) and 82.0 % (95% CrI 65.1 – 93.3), respectively. The sensitivity and specificity of the qPCR method in cats were estimated as 90.1 % (95% CrI 83.5 – 95.6) and 96.2 % (95% CrI 82.2 – 99.8), respectively. The true prevalence of T. gondii infection in feral cats on Phillip Island was estimated as 90.2 % (95% CrI 83.1 – 95.2). Environmental contamination with T. gondii oocysts was assessed. Soil was collected from two sites on the island, Summerland Peninsula and Cape Woolamai, in September and October 2017. A total of 412 soil samples, from 206 sites, was tested. Soil samples were processed to isolate any protozoan organisms present and qPCR was conducted to specifically detect T. gondii DNA. Toxoplasma gondii oocysts were not detected by qPCR in any of the soil samples collected. Additionally, European rabbits, Oryctolagus cuniculus, were used as an indicator species to predict environmental exposure in eastern barred bandicoots. A total of 134 feral rabbits was sampled from August 2016 to November 2017. Overall prevalence of T. gondii in feral rabbits, as determined by qPCR on tissue, was 10.5 % (95% CI 6.3 - 16.8). Toxoplasma gondii infection was not identified in any rabbits collected from the proposed release site for eastern barred bandicoots, the Summerland Peninsula. Sixty-seven eastern barred bandicoots were released onto the Summerland Peninsula in October and November 2017 as part of an assisted colonisation trial. Sixty-one of these bandicoots were tested for prior exposure to T. gondii using the MAT. No animals were seropositive for T. gondii prior to release on to Phillip Island, and the population could be concluded to be free from disease, at a design prevalence of 6 %. Bandicoots were trapped for blood sample collection in November 2017 after approximately three weeks of habitation on the island (n = 23), and again in February 2018 after approximately four months (n = 21). No bandicoots demonstrated seroconversion to T. gondii on the MAT in either November or February. In November, this was sufficient to conclude that the population was free from infection with T. gondii, at a design prevalence of 6 %. However, in February, the sample size was too small to make this conclusion. Mortality data on the bandicoots was limited, due to difficulties associated with placing radio-transmitters on this species. However, one deceased animal was recovered. This animal had no evidence of T. gondii infection based on qPCR on tissues. These findings suggest that while prevalence of T. gondii infection is very high in feral cats on Phillip Island, other factors, such as low cat density at the site, may translate to a low toxoplasmosis risk for eastern barred bandicoots released onto the Summerland Peninsula. Further studies are required to understand the factors contributing to the observed low level of environmental contamination with oocysts. Additionally, future studies should focus on increasing the sensitivity of mortality surveillance in the eastern barred bandicoot population on the Summerland Peninsula. This will enable the collection of more robust mortality data, further elucidating the importance of toxoplasmosis in this population.
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ItemEvaluating effectiveness of a Continuous Glucose Monitoring System (CGMS) in diabetic dogs and cats( 2018)Real-time continuous glucose monitoring systems (CGMS) measure interstitial glucose concentrations, and have been used in the management of diabetes mellitus in people, dogs and cats. The devices are used for up to 72 hours, and provide glucose measurements every 5 minutes, with 288 data points provided in a 24-hour period. This provision of a detailed insight into glycaemic control over a longer period of time than traditional methods of monitoring holds the potential for improved management of diabetes mellitus. The primary aim of this study was to determine if CGMS (using the Guardian™ system) resulted in different clinical decision making compared with monitoring serial blood glucose curves and serum fructosamine concentration in diabetic dogs and cats. Secondary aims were to determine the incidence of nocturnal hypoglycaemia and rebound hyperglycaemia in diabetic dogs and cats. Continuous glucose monitoring and fructosamine measurement were performed in client-owned dogs and cats, both newly and previously diagnosed with diabetes mellitus. A retrospective serial glucose curve was plotted with glucose measurements every 2 hours obtained from the CGMs data. Results of the three monitoring modalities along with historical data (i.e. appetite, thirst, insulin dosage) were collated and a blinded review performed by two board certified small animal internal medicine clinicians. Statistical analysis showed a difference in clinical treatment recommendations for the management of diabetic dogs and cats when using CGMs versus both serial glucose curves and serum fructosamine. Nocturnal hypoglycaemia was seen in 14.6% of diabetic dogs and cats and the 9.8% had episodes of the Somogyi effect.
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ItemEvaluation of tibiotarsal transarticular immobilization techniques and kinematic study of the gastrocnemius muscle-tendon unit in dogs( 2018)The common calcaneal tendon is a complex muscle-tendon system that regulates flexion/extension of the tibiotarsal joint motion as well as the phalanges. Common calcaneal tendon injury is not uncommon, and two types of injuries are recognized: acute or chronic. Acute injuries are usually related to trauma by a sharp object, transecting skin and underlying structures, including tendons. No definitive causes of chronic injury have been identified, but middle age, large-breed dogs are more predisposed to be affected. The treatment of common calcaneal tendon injury consists of debridement, anastomosis and temporary tibiotarsal joint immobilization during tendon healing. Acute traumatic common calcaneal tendon injury is managed in a very similar way for each patient. Several types of immobilization technique have been described, however, no optimal immobilization technique that neutralizes tension in the common calcaneal tendon has been identified. Numerous biomechanical studies of common calcaneal tendon repairs are reported in human and animal models. In human patients, early controlled motion minimizes various complications, resulting in better outcomes. However, there is no gold standard postoperative protocol for veterinary patients. It can be problematic in veterinary patients to control patient activity and have good client and patient compliance. In this research project, six tibiotarsal transarticular fixation methods were evaluated, and a kinematic evaluation of the gastrocnemius tendon was performed at various femorotibial and tibiotarsal joint angle combinations. These data will help inform development of an optimal immobilization method during healing of common calcaneal tendon injury in dogs.
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ItemThe effect of vaccination against Campylobacter on maiden ewe reproduction in Victoria( 2017)Reducing reproductive wastage is important for the Australian sheep industry. Campylobacter fetus fetus and C. jejuni infections in ewes contribute to reproductive wastage through abortions, stillbirths and the birth of small, weak neonates, potentially at greater risk of starvation-mismothering-exposure (SME). A combined vaccine against C. fetus fetus and C. jejuni (Ovilis Campyvax®, MSD Animal Health) is registered in Australia to reduce reproductive wastage due to Campylobacter, but few independent field trials of the vaccine have been conducted in commercial flocks. This study described the effects of Ovilis Campyvax® on maiden ewe reproduction in a randomised controlled field trial on four winter-/spring-lambing Victorian sheep farms. Conception and lamb marking rates were compared amongst nineteen-month-old Merino and Merino-cross ewes randomly allocated to vaccination or control groups at mating on each farm (each n = 211–249/group). Ewes were grazed together from mating until immediately before lambing, when they were set-stocked in treatment groups in matched paddocks. Antibody titres to Campylobacter spp. were measured at mating, mid-gestation pregnancy diagnosis and lamb marking in a subset of ewes. A cross-sectional study of cause of neonatal lamb mortality was also conducted on each farm during lambing. Vaccination had no effect on ewe conception rate (67% to 117% depending on farm). Two of four farms had serological evidence of prior exposure to C. fetus fetus, and variable exposure to this organism occurred during gestation on all farms. Campylobacter jejuni titres were high on all farms at mating, but decreased thereafter. Despite serological evidence of a good response to C. fetus fetus vaccination on all farms, vaccination did not significantly increase lamb marking rates (63% to 100%, depending on farm). The main causes of lamb mortality were dystocia, starvation-mismothering-exposure and predation. There was a suggestion of a difference in the pattern of causes of neonatal lamb mortality between vaccinated and control ewes. The difference was not statistically significant, but corresponded with anecdotal observations made by the flock owners. Additional large scale studies into vaccination and the causes of neonatal lamb mortality are needed to further investigate these observations. Vaccination appeared to prevent Campylobacter-associated neonatal lamb mortality and morbidity on the farm with the greatest exposure to C. fetus fetus. On that farm, 55% of unvaccinated ewes that failed to rear a lamb had ‘high’ (≥ 1:80) C. fetus fetus titres, compared to 0% of ewes that successfully reared a lamb. Additionally, C. fetus fetus was only recovered from necropsied lambs born to unvaccinated ewes. The results demonstrate that ewes can be vaccinated with Ovilis Campyvax® during mating without impacting conception rates. However, the effect of Campylobacter vaccination on reproductive output is complex and multifactorial. Vaccination effects may be obscured by other causes of reproductive loss. Vaccination may reduce the contribution of Campylobacter infections to lamb loss due to SME. However, the dystocia risk in protected ewes may increase depending on ewe nutrition. If this is the case, the nutrition of vaccinated ewes could be managed more economically to obtain the full benefits of vaccination. This is an avenue for future research.
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ItemInfluence of retractor type and position on thoracoscopic-assisted pulmonary surgery in dogs( 2018)Thoracotomy is performed frequently in dogs for the purposes of exploration of the thorax, lung lobectomy, correction of vascular ring anomalies and patent ductus arteriosus, pericardiectomy, thoracic duct ligation, biopsy and resection of mediastinal and pleural masses and removal of foreign bodies from within the pleural space, intrathoracic oesophagus and the lower respiratory tract. Persistent post-thoracotomy pain is reported frequently by human patients characterised by discomfort and altered sensation in the skin adjacent to the surgical site. Multiple surgical techniques have been developed in an attempt to reduce the morbidity of these procedures. Minimally invasive surgical techniques have been a significant step towards this goal. A significant limitation of minimally invasive surgery is the loss of tactile feedback and video-assisted techniques which utilise smaller incision, permitting the introduction of the surgeon’s hand or fingers to the thorax have been developed to overcome these limitations. These techniques have recently been introduced for veterinary patients (Chambers and others 2012, Gower and Mayhew 2011a, Laksito and others 2010b, 2011, Wormser and others 2014). Both traditional and video or thoracoscopic-assisted techniques require the use of tissue retractors, typically placed in an intercostal position. Access is afforded by the forceful retraction of soft tissues such as the intercostal muscles and ribs. Studies in both animals and humans have demonstrated detrimental soft tissue changes associated with such retraction including direct and ischaemic damage to the soft tissues adjacent to the retraction device. The present study examines several aspects of thoracoscopic-assisted pulmonary surgery in dogs including the effect on blood flow in the intercostal musculature and nerve conduction in the intercostal nerves associated with two different retraction modalities. Additionally, the influence of assisted port location and thoracic conformation on access to and delivery of pulmonary tissues for the purposes of complete or partial lung lobectomy was assessed. Force, contact area and pressure were compared and related to intramuscular pressure, perfusion, oxygen indices, nerve conduction and histologic change in dogs undergoing simultaneous controlled thoracotomy incisions. The novel retractor resulted in lower pressure and improved oxygenation in adjacent tissues compared to mechanical retraction which may translate clinically into reduced post-operative morbidity. For all lobes except the accessory lung lobe, the exposure provided by the reported thoracoscopic-assisted approach was considered adequate for complete or partial lung lobectomy. The cranial and caudal portions of the left cranial lung lobe and the right middle lung lobe were best exposed with the assisted portal in the middle dorso-ventral third of the thoracic wall at the fourth intercostal space of the ipsilateral side in both breeds. The right cranial lung lobe was best exposed with the assisted portal positioned in the middle third of the fourth intercostal space in kelpies but the sixth intercostal space in greyhounds. The right caudal lung lobe was best exposed when the assisted portal was positioned in the ventral third of the fourth intercostal space in the kelpie but in the middle third of the sixth intercostal space in the greyhound. The described thoracoscopic-assisted technique is a practical method for exposure of the lobes of the lung, other than the accessory lobe, for partial lung lobectomy in the dog. The portals described also allow the introduction of a linear stapler for the purpose of complete lung lobectomy.